Mitochondrial ATP Synthase Catalytic Mechanism: A Novel Visual Comparative Structural Approach Emphasizes Pivotal Roles for Mg²⁺ and P-Loop Residues in Making ATP

Abstract

The mitochondrial ATP synthase (F(o)F(1)) is one of the most abundant, important, and complex enzymes found in animals and humans. In earlier studies, we used the photosensitive phosphate analogue vanadate (V(i)) to study the enzyme's mechanism in the transition state. Significantly, these studies showed that Mg(2+) plays an important role in transition state formation during ATP synthesis. Additionally, in both MgADP·V(i)-F(1) and MgV(i)-F(1) complexes, photoactivation of orthovanadate (V(i)) induced cleavage at the third residue within the P-loop (GGAGVGKT), i.e., βA158, suggesting its proximity to the γ-phosphate during transition state formation. However, despite our recent release of the F(1)-ATPase structure containing V(i), the structural details regarding the role of Mg(2+) have remained elusive. Therefore, in this study, we sought to improve our understanding of the essential role of Mg(2+) during transition state formation. We utilized Protein Data Bank structural data representing different conformational intermediates of key steps in ATP synthesis to assemble a database of positional relationships between landmark residues of the catalytic site and the bound ligand. Applying novel bioinformatics methods, we combined the resulting interatomic spatial data with an animated model of the catalytic site to visualize the exact nature of the changes in these positional relationships during ATP synthesis. The results of these studies reported here show that the absence of Mg(2+) results in migration of inorganic phosphate (P(i)) from βA158 to a more medial position in the P-loop binding pocket, thereby disrupting essential placement and orientation of the P(i) needed to form the transition state structure and therefore MgATP.

Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes

“…It uses four structures, including the ATPase·Mg·ADP·AlF 4 – complex (PDB: 1h8e ; Fig. 20) [108].

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“…However, recent advances in computational methodology have cast a shadow on earlier methods, where the energy error might easily lie in the range of 2–10 kcal mol −1 , with error spreading as large as 30 kcal mol −1 [78, 114, 115]. Current protocols enable a much larger number of heavy atoms to be embraced in the QM zone, leading to computer results that hinge on geometry of the TS and the H-bond network that it embraces [44, 108, 111]. Such analyses have identified the propensity of nucleotide analogs, particularly β,γ-iminoATP, β,γ-methyleneATP, and their GTP counterparts, to deliver NACs for phosphoryl transfer processes, which is now recognized in recent computational studies as capable of generating small but highly significant conformational changes in kinases and GTPases [44, 108].…”

“…Current protocols enable a much larger number of heavy atoms to be embraced in the QM zone, leading to computer results that hinge on geometry of the TS and the H-bond network that it embraces [44, 108, 111]. Such analyses have identified the propensity of nucleotide analogs, particularly β,γ-iminoATP, β,γ-methyleneATP, and their GTP counterparts, to deliver NACs for phosphoryl transfer processes, which is now recognized in recent computational studies as capable of generating small but highly significant conformational changes in kinases and GTPases [44, 108]. Lastly, the belief that enzymes work by optimizing reaction mechanisms that work slowly in solution, as Knowles put it “Not different, Just better” [116], is proving to be wide of the mark for phosphate reactions.…”

Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes

“…However, recent advances in computational methodology have cast a shadow on earlier methods, where the energy error might easily lie in the range of 2-10 kcal mol -1 , with error spreading as large as 30 kcal mol -1 [78,114,115]. Current protocols enable a much larger number of heavy atoms to be embraced in the QM zone, leading to computer results that hinge on geometry of the TS and the H-bond network that it embraces [44,108,111]. Such analyses have identified the propensity of nucleotide analogs, particularly b,c-iminoATP, b,cmethyleneATP, and their GTP counterparts, to deliver NACs for phosphoryl transfer processes, which is now recognized in recent computational studies as capable of generating small but highly significant conformational changes in kinases and GTPases [44,108].…”

“…Current protocols enable a much larger number of heavy atoms to be embraced in the QM zone, leading to computer results that hinge on geometry of the TS and the H-bond network that it embraces [44,108,111]. Such analyses have identified the propensity of nucleotide analogs, particularly b,c-iminoATP, b,cmethyleneATP, and their GTP counterparts, to deliver NACs for phosphoryl transfer processes, which is now recognized in recent computational studies as capable of generating small but highly significant conformational changes in kinases and GTPases [44,108]. Lastly, the belief that enzymes work by optimizing reaction mechanisms that work slowly in solution, as Knowles put it ''Not different, Just better'' [116], is proving to be wide of the mark for phosphate reactions.…”